One of the major unanswered questions in vascular biology is the process of smooth muscle cell (SMC) differentiation. SMCs are a highly specialized cell type yet exhibit a plasticity in which there is de-differentiation or phenotypic modulation in the context of vascular injury and intimal lesion formation. It is well established that embryonic stem (ES) cells can be utilized as an effective tool for studying the molecular mechanisms governing cell differentiation during development. During ES cell differentiation to specific cell lineage, each stage progenitor of early development will recapitulate, that is a unique advantage of ES cells subject to research model and that we can study cell differentiation dynamically in vitro. Recently, we have established a protocol that mouse ES cells can be directly differentiated to SMC with a high efficiency, which enable us to study molecular mechanisms of SMC differentiation. microRNAs (miR) are a group of recently discovered small non-coding RNAs. The functions of miR have been identified in both normal physiological and pathological conditions, including metabolism, proliferation, migration, cell death, cell differentiation and development. By establishing SMC differentiation from ES cells, we found that miR-1 is up-regulated, whereas miR-141 is downregulated during the SMC differentiation. The proposed studies will test the central hypothesis that miR-1 and miR-141 act as novel critical determinants of SMC differentiation utilizing gain- and loss-of- function approaches. Specifically, we will: 1) Determine the role of miR-1 in SMC Differentiation;2) Determine the role of miR-141 in SMC Differentiation;and 3) Determine the Role of miR-1 and miR-141 in Vascular Lesion Formation These proposed studies will establish a unique model system for characterizing the mediator roles of miR-1 and miR-141 as novel determinants of SMC differentiation both in vitro and in vivo. This proposed grant is the initial phase of our long-term objective to define the role of SMC differentiation in vascular diseases. Ultimately, our studies will lead us to discover novel SMC differentiation regulators and advance our understanding of SMC differentiation and vascular diseases. PUBLIC HEALTH RELEVANCE: One of the major unanswered questions in vascular biology is the process of SMC differentiation. Recently, we have established a protocol that mouse ES cells can be directly differentiated to SMC with a high efficiency, which enable us to study molecular mechanisms of SMC differentiation. microRNAs (miR) are a group of recently discovered small non-coding RNAs. The functions of miR have been identified in both normal physiological and pathological conditions. This proposal will establish a unique model system for characterizing the mediator roles of miR-1 and miR-141 as novel determinants of SMC differentiation both in vitro and in vivo. This proposed grant is the initial phase of our long-term objective to define the role of SMC differentiation in vascular diseases. Ultimately, our studies will lead us to discover novel SMC differentiation regulators and advance our understanding of SMC differentiation and vascular diseases.